Evaluation of different probiotics on growth, body composition, antioxidant capacity, and histoarchitecture of Mugil capito

We investigated the dietary effects of the single application of Saccharomyces cerevisiae, Lactobacillus bulgaricus, and their combination on growth, proximate composition of whole fish body, antioxidant defense, and histoarchitecture of hapa-reared Mugil capito. Healthy fish (Fish weighed = 10.30 ± 0.10 g at first) were randomly allocated into 4 equal groups, each with three replicates. These groups were designed as follows: (1) a group fed a basal diet without probiotics (control), (2) a group fed a diet containing S. cerevisiae (4 g/kg diet), (3) a group fed a diet containing L. bulgaricus (2 g/kg diet), and (4) the last group fed a diet containing a combination of both, all for a duration of 60 days. Probiotic-treated groups showed significantly better growth and nutrition utilization than the control group. Significant differences were observed in the crude fat and crude protein contents among the groups, with the combination group exhibiting the highest levels. However, there were no significant variations in ash content across all groups. The highest hepatic antioxidant capacity (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) enzyme activities) was observed in the combination group. Thiobarbituric acid reactive substance (TBARS) concentrations were decreased significantly in all probiotic groups, suggesting improved oxidative stress resilience in these groups. The histomorphological analysis of the hepatopancreatic tissues revealed well-arranged parenchyma, increased glycogen storage, and melanomacrophage centers in probiotic-treated groups, particularly the combined probiotics group. Furthermore, the probiotic supplementation improved the histoarchitecture of the intestinal villi compared to the control group. To put it briefly, combined dietary administration of these probiotics improved growth, body composition, antioxidant defenses, and hepatic and intestinal health in hapa-reared M. capito, highlighting their promising role in promoting welfare and productivity.


Fish acquisition and acclimation
One hundred and twenty healthy M. capito were sourced from a private farm located in the district of Kafr Elsheikh, Egypt.These fish were housed and nurtured in four 500-L fiberglass aquariums, each equipped with efficient aeration systems to maintain water quality.To ensure their successful acclimatization to the new environment, fish were allowed to acclimate in these tanks for 2 weeks.During this time, fish were provided with ad libitum feeding, using a nutritionally balanced commercial diet sourced from Aller Aqua Co., Egypt.

Probiotics used and diet preparation
The S. cerevisiae cell wall extract (Megamos®) utilized in this research was commercially purchased from Biobridtech (USA) by a Local Agent supplier.It was added to the baseline diet at a rate of 4 g/kg diet, as recommended in the prior studies conducted by Islam et al. 31 and Fadl et al. 32 .Lactobacillus bulgaricus (Batch No. EL1010322) is a freeze-dried powder purchased commercially from APEX BIOTECHNOL in Indore, Madhya Pradesh, India, via Free Trade Egypt Company in Alexandria, Egypt.This strain of L. bulgaricus is marketed as a probiotic foodgrade dietary supplement, boasting a potency of 21.5 × 10 9 CFU/g, which underscores its high viability and total cell count.It was incorporated into the basal diet at a supplementation dose of 2 g/kg diet.The probiotic strains were maintained at − 20 °C until it was needed for experimentation.To assess the comparative effectiveness of single-strain and their mixture in enhancing the growth and health status of M. capito, we designed four distinct diets.The control group received only the baseline diet without any additional supplements (refer to Table S1 in the Supplemental Material).The second diet involved the supplementation of the basal diet with S. cerevisiae (4 g/kg diet).For the third diet, L. bulgaricus (2 g/kg diet) was incorporated into the basal diet.The fourth diet combined both supplements (S. cerevisiae, 4 g/kg diet + L. bulgaricus, 2 g/kg diet).The test probiotic doses were mixed into a powdered nutrient base to ensure uniform distribution.Water was added to the feed ingredients to form a workable paste, which was then processed through a food pelleting machine to create uniform 2-mm pellets.These pellets were air-dried and stored in airtight plastic bags at − 20 °C until required.The diets were formulated to be isonitrogenous and isocaloric, meeting the nutritional requirements for rearing fish as per the guidelines of NRC 33 .

Samples collection
After the feeding trial, the basal and test diets were withheld from the fish in all hapas for one day before sampling.Initially, the total number of fish per hapa, individual body weight, and total weight in each hapa were determined.Clove oil at a concentration of 20 mg/L was used for fish anesthesia.Nine fish (three from each hapa) were randomly selected from each group, euthanized with an overdose of clove oil (40 mg/L), and their livers and intestines were then aseptically extracted to collect samples.The samples were immediately rinsed with sterile cold phosphate-buffered saline (PBS) solution (pH ~ 7.4) and fixed in a 10% buffered formalin solution for 2 days to facilitate histological examination.Additionally, a set of liver samples (nine per group) were taken for the preparation of tissue homogenates to assess hepatic oxidative stress biomarkers.Another set of 3 fish per group was sampled to evaluate the chemical composition of the fish's whole body.

Growth parameters and measurements
The following formulas were utilized to calculate growth, feed utilization parameters, and survival rate.
Weight gain (WG): Specific growth rate (SGR): where "Ln" represents the natural logarithm and "days" is the number of days over which the growth occurred.Feed intake (FI): Feed conversion ratio (FCR): Protein efficiency ratio (PER): Protein productive value (PPV): Energy retention (ER): Survival rate (SR):

Chemical composition analysis
The moisture, crude protein (CP), crude lipids (CL), and ash content were conducted following the guidelines outlined by AOAC 34 .

Hepatic antioxidant capacity analysis
Utilizing a Teflon-coated mechanical homogenizer, 0.1 g of hepatic tissue was homogenized in 0.9 mL of PBS solution.The samples were subsequently centrifuged for 15 min at 3500×g to obtain the supernatant.Hepatic oxidative stress biomarkers, including SOD, CAT, and GPX enzyme activities, were assessed using commercial ELISA kits (SOD kit: CSB-E15929Fh, CAT kit: CSB-E15928Fh, GPX kit: CSB-E15930Fh) obtained from Cusabio Biotech Company, Ltd. (Wuhan, China), following the guidelines provided by the manufacturer.The concentration of hepatic Malondialdehyde (MDA) was determined using commercial kits from Biodiagnostic Co. (Giza, Egypt).MDA concentrations, indicative of lipid peroxidation, in the test samples were determined using a spectrophotometric technique as detailed by Ke et al. 35 based on the assessment of thiobarbituric acid reactive substance (TBARS) in fish tissues.Furthermore, following the methodology outlined by Benzie and Strain 36 , an antioxidant power experiment was conducted at an optical density (OD) of 593 nm.The livers and intestines preserved in formalin were processed using the paraffin embedding technique, following the guidelines outlined in Bancroft and Gamble 37 .Tissue slices were stained with hematoxylin and eosin, and observed using an Olympus BX50/BXFLA microscope (Japan).

Statistical analysis
The data were analyzed using one-way ANOVA followed by post hoc analysis with Duncan's Multiple Range Test.The findings were presented as mean values with standard error of the mean (SEM).Statistical significance was considered when p < 0.05.SPSS software was utilized for the statistical analysis.

Results
Growth performance, nutrient utilization, and survival rate of M. capito In Table 1, several indicators exhibited notable differences among the treatments.The combination treatment group exhibited the highest FBW than the rest of the treatments.This trend was consistent with WG, SGR, and FI, where the combination group outperformed the other groups, indicating significant differences (p < 0.05).Additionally, the PER was significantly higher in the combination group (p < 0.05), compared to the control group.Furthermore, the PPV demonstrated a similar trend, with the combination group surpassing other groups significantly (p < 0.05).Finally, the highest protein efficiency was observed in the combination group, evident from the elevated E. value compared to other treatments.Notably, SR was consistent across all groups, remaining at 100%.

Proximate body composition of M. capito
The results indicate significant differences among the treatment groups for various parameters in Table 2. Moisture content (%) was highest in the control group, while the combination of S. cerevisiae and L. bulgaricus resulted in the lowest moisture content.CP showed significant variation (p < 0.05), with the highest value observed in the combination group compared to the control.Similarly, CL content was significantly different across treatments (p < 0.05), with the combination group exhibiting the highest value.In contrast, ash content showed no significant differences among the groups (p > 0.05).

Histological observations of the intestinal and hepatic in M. capito
The histological structure of M. capito intestine showed intact structures of the intestinal wall and intestinal villi of all segments in all experimental groups (Fig. 1A-C).The histological appearance of the intestinal villi showed a significant enrichment with supplemented S. cerevisiae or L. bulgaricus (Fig. 1B2-C4).Interestingly, L. bulgaricus gave a slightly better histomorphometry of the intestine than S. cerevisiae without significant differences from the combination of both supplemented elements.The histopathological examination of the liver in the control group revealed normal hepatic parenchyma and intact hepatocytes arranged in hepatic cords that were separated by slightly congested blood sinusoids and central veins (Fig. 2A).L. bulgaricus-supplemented groups as well as S. cerevisiae with L. bulgaricus (Fig. 2C,D) groups showed more improved construction of the hepatic parenchyma represented in increased glycogen storage with pigmented melanomacrophage centers near the hepatic central veins while S. cerevisiae-supplemented group (Fig. 2B) showed less improvement.

Discussion
A mounting body of research has underscored the capacity of probiotics to enhance growth performance, feed utilization, proximate body composition, antioxidant activity, and hepatic health 11,38 .These beneficial outcomes are often attributed to the administration of single-species probiotic treatments, as elucidated in numerous studies 16,22 .However, scant attention has been devoted to investigating the benefits arising from the concurrent introduction of distinct probiotic species into fish diets 39,40 .It has been hypothesized that a multispecies probiotic combination could potentially offer more advantages compared to single-strain probiotics when administered to fish 24 .This hypothesis is thought to stem from synergistic interactions among probiotic strains, as supported by studies conducted by Abdel-Latif et al. 24 and Wang et al. 41 .
The growth performance and nutritional outcomes observed in this study indicate the potential benefits of probiotic supplementation in hapa-reared M. capito.These findings follow previous studies examining the effects of probiotics on various fish species such as red sea bream (Pagrus major) 42 , gilthead sea bream (Sparus aurata) 43 , and olive flounder (Paralichthys olivaceus) 44 .This enhancement in growth parameters can be attributed to the probiotics' ability to improve nutrient utilization and digestion efficiency, as reported in previous studies on fish 45,46 .PER and PPV are important indicators of the nutritional value of the diet, and their significant improvement in the combination group suggests that S. cerevisiae and L. bulgaricus supplementation enhances the utilization of dietary protein.This aligns with research on other fish species, where probiotics have been shown to enhance protein utilization and improve the overall nutritional quality of the diet 4 .The elevated E.R. value in the combination group further supports the notion that probiotic supplementation can optimize protein utilization in M. capito.To the best of our knowledge, this study marks the inaugural instance of dietary supplementation with S. cerevisiae and/or L. bulgaricus yielding improvements in the growth and feed utilization of M. capito.The fish survival remained consistently high across all treatment groups at 100%.This finding suggests that the probiotic supplements did not negatively impact fish.This aligns with previous studies that have reported the safety and non-toxic nature of probiotic supplementation in fish [47][48][49] .
In our current investigation, the CP and CL levels were increased in the whole body of fish-fed diets supplemented with S. cerevisiae and/or L. bulgaricus.These findings suggest that these probiotics have led to enhanced utilization of both protein and lipids.These results are consistent with prior research, as proposed by Dawood et al. 50and Mohapatra et al. 51 .
Probiotics have a potential efficacy in diminishing oxidative stress among aquatic organisms, including fish and shellfish 52,53 .When animals are subjected to stress, it can trigger an increase in the levels of reactive oxygen Table 3. Impacts of probiotic supplements (S. cerevisiae, L. bulgaricus, and their combination) on hepatic antioxidant activity of M. capito over a 60-day trial.All values are presented as mean ± SE, n = 3. SOD superoxide dismutase, CAT catalase, GPX glutathione peroxidase, TBARS thiobarbituric acid reactive substance.The alphabetical superscripts within the values denote significant differences (p < 0.05) between various treatments within each row.***Combination = S. cerevisiae + L. bulgaricus.www.nature.com/scientificreports/species (ROS).These ROS play a vital role as chemical signaling molecules within the organism.However, oxidative stress occurs when there is an imbalance between ROS/RNS (reactive nitrogen species) and antioxidants, leading to cell damage through processes such as lipid peroxidation.In response, the fish's antioxidant defense system becomes activated, combatting the generated free radicals by releasing antioxidant enzymes like SOD, CAT, and GPX.These antioxidant enzymes play pivotal roles in mitigating oxidative stress in fish.These enzymes function to endorse the regular redox equilibrium 54,55 .The results of this study reveal noteworthy variations in antioxidant enzyme activity and oxidative stress levels across the investigated groups, shedding light on the potential benefits of concomitant supplementation with two distinct probiotics.When compared to the control group, both S. cerevisiae and L. bulgaricus individually demonstrated an increase in the activities of SOD, CAT, and GPX.Notably, the combination group exhibited the most remarkable enhancement in these enzymatic activities.These findings align with previous investigations into the antioxidant effects of probiotics (e.g. 56,57).The TBARS levels, as an indicator of oxidative stress 58 , followed an opposing trend.The decrease of TBARS in probiotic groups suggests that the combination of S. cerevisiae and L. bulgaricus exerts a potent antioxidative effect, potentially mitigating oxidative stress-induced damage more effectively than individual probiotic supplementation.Previous research also provides supporting evidence for the role of probiotics possessing antioxidant properties in enhancing intracellular antioxidant enzymes.For instance, Abarike et al. 59 observed increased SOD and GPX activity in the serum of Labeo rohita and Oreochromis mossambicus when fed a diet containing 10 8 CFU/g of L. plantarum and 10 7 CFU/g of Bacillus licheniformis, respectively.It was proposed that these probiotics exert their antioxidant effects through mechanisms such as ion chelation, reduction of reactive oxygen metabolites, prevention of oxidant compound production, reduction of ascorbate autoxidation, and scavenging of ROS, as suggested by Amaretti et al. 60 and Naderi Farsani et al. 57 .Probiotics exert antioxidant effects and enhance fish health through a multifaceted approach involving modulation of gut microbiota, production of antioxidant compounds, reduction of inflammation, enhancement of immune function, improvement of antioxidant enzyme activity, regulation of gut barrier function, detoxification of metabolic byproducts, and enhanced nutrient absorption 61 .These mechanisms collectively contribute to mitigating oxidative stress and promoting overall fish health.Probiotics enhance the diversity and balance of gut microbiota, leading to improved gut health and nutrient absorption 62 .Additionally, certain strains of probiotics produce antioxidant compounds such as glutathione, superoxide dismutase, and catalase, which scavenge free radicals and reduce oxidative stress 63 .Furthermore, probiotics modulate immune responses, reduce inflammation, enhance antioxidant enzyme activity, and regulate gut barrier function, collectively contributing to improved fish health 64,65 .Nonetheless, additional research is warranted to elucidate the underlying mechanisms and evaluate the effects of these probiotics under induced stress conditions, such as overcrowding or transportation.Conducting pro-oxidant challenge studies would allow for a comprehensive assessment of their impact in such scenarios.
The maturation and functionality of internal organs, particularly the intestine and liver, exert a profound impact on the growth of fish, bolstering their resilience against diseases and stressors while enhancing their efficiency in utilizing feed resources 66 .In the present study, the histological analysis of M. capito treated with a combination of probiotics exhibited notable improvements in both the intestinal and hepatic tissues compared to the control group.The histological examination of the M. capito intestine across all experimental groups revealed that the intestinal wall and villi remained structurally intact.This finding suggests that the dietary supplements, whether S. cerevisiae or L. bulgaricus, did not cause any significant damage to the intestinal structure.Both supplements appeared to enrich the histomorphometry of the intestinal villi.Previous research has highlighted the beneficial effects of probiotics such as L. bulgaricus on gut health.Probiotics can enhance the integrity of the intestinal mucosal barrier, improve the composition of gut microbiota, and promote the growth of beneficial enterocytes and goblet cells [66][67][68] .The enrichment observed in this study aligns with these known effects of probiotics.Interestingly, the combination of both S. cerevisiae and L. bulgaricus showed a similar effect on histomorphometry compared to L. bulgaricus alone, implying that L. bulgaricus may play a predominant role in enhancing intestinal structure.It is essential to note that while the histological structure appeared to improve with these supplements, additional functional studies would be required to ascertain their impact on nutrient absorption and overall gut health.
The groups supplemented with L. bulgaricus or the combination of S. cerevisiae and L. bulgaricus demonstrated an improvement in the construction of hepatic parenchyma.This was characterized by increased glycogen storage and the presence of pigmented melanomacrophage centers near the central veins.Glycogen storage is indicative of the liver's ability to store glucose and maintain blood sugar levels 69,70 .The presence of melanomacrophage centers suggests an enhanced immune response in the liver 71,72 .This could be attributed to the immunomodulatory properties of probiotics, which have been reported to have anti-inflammatory effects and support liver health 73,74 .Probiotics have been associated with improved liver function, reduced inflammation, and protection against liver diseases 75,76 .Taken together, the histological analysis of the M. capito intestine and liver suggests that dietary supplementation with S. cerevisiae and L. bulgaricus has a positive impact on the structural integrity of these organs.L. bulgaricus, in particular, appears to play a significant role in enhancing both intestinal and hepatic histomorphometry.These findings are consistent with existing research on the beneficial effects of probiotics on gut and liver health.However, further studies are needed to elucidate the underlying mechanisms and assess the functional implications of these histological improvements.

Conclusion
In summary, this study underscores the beneficial impacts of orally administering S. cerevisiae and/or L. bulgaricus probiotics on various aspects of hapa-reared M. capito, including growth, feed utilization, body composition, hepatic antioxidant capacity, and histoarchitectural changes.Notably, the combination of these probiotics in a multispecies formulation exhibited superior efficacy compared to individual supplementation.These findings suggest mixing different probiotics to enhance fish performance and overall health.Nonetheless, further studies are warranted to elucidate the precise mechanisms involved and to assess the long-term effects on fish health and welfare.

Figure 2 .
Figure 2. The photograph shows the histological structure of M. capito liver in the control group (A) as well as other treated groups by S. cerevisiae (B), L. bulgaricus (C), and their combination (D).The construction of the liver in the control fish presented a normal appearance of hepatocytes (A), arranged in hepatic cords that were separated by slightly congested blood sinusoids and central veins.S. cerevisiae and L. bulgaricus-supplemented groups prompted enhanced hepatic parenchyma with increased glycogen storage, and melanomacrophage centers (green arrowhead), particularly in the mixture group.Stain H&E.Bar 100 µm.

Table 1 .
Impacts of probiotic supplements (S. cerevisiae, L. bulgaricus, and their combination) on growth, nutrient utilization, and survival of M. capito over a 60-day trial.All values are presented as mean ± SE, n = 3. IBW initial body weight, FBW final body weight, WG weight gain, SGR specific growth rate, FI feed intake, FCR feed conversion ratio, PER protein efficiency ratio, PPV protein protective value, ER energy retention, SR survival rate.The alphabetical superscripts within the values denote significant differences (p < 0.05) between various treatments within each row.***Combination = S. cerevisiae + L. bulgaricus.

Table 2 .
ImpactsThe results of the study demonstrate significant variations in antioxidant enzyme activity and oxidative stress biomarkers among the tested groups, as illustrated in Table3.It was found that S. cerevisiae and L. bulgaricus individually showed increased SOD, CAT, and GPX activities in comparison to the control group.The combination group exhibited the highest antioxidant enzymatic activities (p < 0.05).Conversely, TBARS assay results, an indicator of oxidative stress, displayed the opposite trend.The control group had the highest TBARS levels, while S. cerevisiae, L. bulgaricus, and the combination group exhibited lower TBARS levels, with the combination group showing the most pronounced reduction (p < 0.05).